Blood feeding mosquitoes use sensitively tuned chemosensory systems to locate their vertebrate hosts. At locations far from a target host, anthropophilic mosquitoes predominantly rely on their sense of smell to follow plumes of volatile host emissions emanating from the human body, which include skin odor and carbon dioxide (CO2). In the yellow fever mosquito Aedes aegypti, CO2 is sensed by specialized populations of olfactory sensory neurons (OSNs) housed within capitate peg sensilla on the ventral surface of the maxillary palps. In each CO2-sensitive neuron, three gustatory receptors (Grs) named Gr1, Gr2 and Gr3, are hypothesized to interact to form a CO2 receptor complex mediating detection of this volatile gas, as well as structurally diverse ligands including certain heterocyclics, ketones and alcohols. Whether these three Gr subunits are all functionally required for CO2 receptor complex formation and the broad tuning responses of this neuron to this gas and other odorants, or whether additional signaling cofactors are involved is unknown. Here, we propose to apply integrative methods to functionally define the molecular architecture of the mosquito CO2 receptor complex. We will initially apply single-sensillum electrophysiology to functionally probe responses of Ae. aegypti Gr1, 2 and 3 null mutants to CO2 and a select panel of volatile agonists and antagonists of this receptor complex. Using CRISPR-Cas9 genome editing, we will additionally insert small-epitope tags onto the N? and C?terminal ends of Gr1, 2 and 3 to evaluate the ability of each of these individual Grs to traffic independently from the neuronal cell bodies to the lamellate cpA dendrite using immunohistochemistry. These approaches will reveal Gr subunits that may influence the odor tuning sensitivity or specificity of the complex, or those that may serve as co-receptors for transport of ligand-binding subunits to the sensory membrane. To evaluate a specific role for carbonic anhydrases (CAHs) in CO2 receptor complex function, which we hypothesize may catalyze the conversion of CO2 inside the aqueous sensillar lymph to bicarbonate ions and protons to act as receptor ligands, we will ablate target CAHs expressed in the maxillary palps using genome- editing and pharmacological manipulations. We will further highlight the anatomy of carbonic anhydrase expressing cells in the mosquito olfactory system using transgenic tracing methods to query their potential role in gas detection. CO2 is one of the most universal and important components of odor-baited traps used for surveillance and control of hematophagous insects including mosquito disease vectors. An improved understanding of the molecular architecture of the broadly conserved CO2 receptor complex may facilitate the development of efficient heterologous expression systems that reconstitute the mosquito CO2 receptor complex in vitro. Such an advance may aid in the rapid identification of novel volatile compounds mimicking or antagonizing the effects of CO2 for applied use in vector-borne disease control strategies.